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| United States Patent |
5,139,777
|
|
Ott
, et al.
|
August 18, 1992
|
Composition and method for improving the efficiency of ruminant feed
utilization
Abstract
The invention relates to a composition for improving the efficiency of
ruminant feed utilization, which comprises as active ingredient one or
more microbial cultures, capable of adjusting the weight ratio of acetic
acid to propionic acid to an optimum value, preferably to 1.5-4.0:1, and
of growing in the rumen and persisting there at least for 60 days,
optionally in admixture with carriers, diluent, preserving agents
conventionally used in animal husbandry and nutritive and/or other
substances conventionally administered to ruminants.
According to another aspect of the invention there is provided a process
for the preparation of microbial cultures used as active ingredient in the
above composition.
The invention further relates to a process for the preparation and use of
said compositions.
| Inventors:
|
Ott; Istvan (Budapest, HU);
Szentmihalyi; Sandor (Budapest, HU);
Seregi; Janos (Budapest, HU);
Lang; Tibor (Budapest, HU);
Dohy; Janos (Budapest, HU);
Moravcsik; Imre (Budapest, HU);
Kiss; Gyorgy B. (Szeged, HU)
|
| Assignee:
|
Richter Cedeon Vegveszeti (Budapest, HU)
|
| Appl. No.:
|
351295 |
| Filed:
|
May 10, 1989 |
Foreign Application Priority Data
| Current U.S. Class: |
424/93.4; 426/61; 435/38; 435/252.1; 435/471 |
| Intern'l Class: |
A61K 035/74; C12N 015/01; C12Q 001/10; C12R 001/01 |
| Field of Search: |
424/92,93,9
426/2,61,71
435/172.3,253,29,30,34,38,6,42
935/63,72,73,74,75
|
References Cited
U.S. Patent Documents
| 3857971 | Dec., 1974 | Abdo et al. | 426/2.
|
| 3932670 | Jan., 1976 | Sakurai | 426/2.
|
| 3956482 | May., 1976 | Hahn et al. | 435/93.
|
| 4503155 | Mar., 1985 | Miller et al. | 435/172.
|
Primary Examiner: Elmore; Carolyn
Assistant Examiner: Knode; Marian C.
Attorney, Agent or Firm: Keil & Weinkauf
Parent Case Text
This application is a continuation-in-part of application Ser. No. 764,329,
filed on Aug. 12, 1985, now abandoned.
Claims
We claim:
1. A composition for improving the efficiency of ruminant feed utilization
comprising, as the active component, an acetic acid or propionic acid
producing microorganism capable of adjusting the weight ratio of acetic
acid to propionic acid to a value of 1.5 to 4.0:1 and of growing in the
rumen and persisting there at least 60 days wherein the microorganism is a
member selected from the group consisting of Propionibacterium genus
deposit No. NCAIM B(P) 000287, Viellonella genus deposit No. NCAIM B(P)
000288 and Bifidobacteria genus deposit No. NCAIM B(P) 000289, the
deposited numbers being of the National Collection of Agricultural and
Industrial Microorganisms, Budapest, Hungary and a carrier therefor.
2. The composition of claim 1 which additionally comprises a a diluent,
preservative, nutrient or mixtures thereof.
3. The composition of claim 1 which comprises at most 95% by weight of
active ingredient.
4. The composition of claim 1 which comprises a microorganism capable of
adjusting the acetic acid to propionic acid ratio to 2.0-3.5:1.
5. The composition of claim 1 in the form of a microorganism past,
lyophilizate or suspension.
6. A process for the preparation of a composition for improving the
efficiency of ruminent feed utilization, said composition containing an
acetic acid or propionic acid producing microorganism capable of adjusting
the weight ratio of acetic acid to propionic acid to a value of 1.5 to
4.0:1 and of growing in the rumen and persisting there at least 60 days,
wherein the microorganism is a member selected from the group consisting
of Propionibacterium genus deposit No. NCAIM B(P) 000287, Viellonella
genus deposit No. NCAIM B(P) 000288 and Bifidobacteria genus deposit No.
NCAIM B(P) 000289, the deposit numbers being of the National Collection of
Agricultural and Industrial Microorganisms, Budapest, Hungary, comprising
the following steps:
(a) taking samples of microorganisms from the rumen of animals fed on a
given feedstuff,
(b) testing the microorganisms taken from the rumen for the capacity to
product volatile fatty acids in vitro,
(c) selecting microorganisms having the capacity to produce acetic acid or
propionic acid and which are capable of adjusting the weight ratio of
acetic acid to propionic acid to a value of 1.5 to 4.0:1,
(d) cultivating the microorganisms selected in step (c) in media
containing, as the carbon or nitrogen source, the same feedstuff as was
used to feed the animals from which the samples were taken in step (a),
(e) genetically labelling the microbial culture of step (d) to produce
genetically marked strains,
(f) cultivating the genetically marked strains in the same media used in
step (d) to produce genetically marked cultures of microorganisms.
(g) introducing the genetically marked cultures into the rumen of animals
fed on the same feedstuff contained in the media used to cultivate the
microorganisms in step (d),
(h) taking samples of microorganisms from the rumen of the animals in which
the genetically marked cultures were introduced in step (g) at least 60
after the introduction thereof,
(i) selecting the genetically marked strains from the sample taken in step
(h),
(j) cultivating the genetically marked strains selected in step (i) on the
same feedstuff used in step (d) to produce genetically marked cultures of
microorganisms, identified by the above-stated deposited numbers, which
produce acetic acid or propionic acid and which are capable of adjusting
the weight ratio of acetic acid to propionic acid to a value of 1.5 to
4.0:1 and of growing in the rumen and persisting thereat least 60 days,
and
(k) mixing the microbial cultures of step (j) with a carrier, diluent,
preservatives or nutrients.
7. The process of claim 6 in which antibiotic resistance is used as genetic
marker.
8. The process of claim 6 in which microorganisms are isolated from the
rumen of animals fed on a cellulose-containing feedstuff, a
starch-containing feedstuff or a monosaccharide-containing feedstuff,
disaccharide-containing feedstuff or mixture thereof and the genetically
marked strains are introduced into the rumen of animals fed on the same
feedstuff.
9. A process for improving the efficiency of ruminant feed utilization
comprising orally administering to a ruminant animal an effective amount
of an acetic acid or propionic acid producing microorganism capable of
adjusting the acetic acid to propionic acid ratio in the rumen to a value
of 1.5-4.0:01 and of growing in the rumen and persisting there at least 60
days wherein the microorganism is a member selected from the group
consisting of Propionibacterium genus deposit No. NCAIM B(P) 000287,
Viellonella genus deposit No. NCAIM B(P) 000288 and Bifidobacteria genus
deposit No. NCAIM B(P) 000289, the deposit numbers being of the National
Collection of Agricultural and Industrial Microorganisms, Budapest,
Hungary.
Description
The invention relates to a composition for improving the efficiency of
ruminant feed utilization. More particularly, the invention concerns a
composition which comprises as active ingredient one or more microbial
cultures, capable of adjusting the weight ratio of acetic acid to
propionic acid to an optimum value, preferably to 1.5-4.0:1, and of
growing in the rumen and persisting there at least 60 days, optionally in
admixture with carriers, diluents, preserving agents conventionally used
in animal husbandry and nutrition and/or other substances conventionally
administered to ruminants. The invention further relates to the
preparation of microbial cultures used as active ingredient in the above
compositions and to the application of said compositions.
Ruminants possessing composite stomach, as sheep (Ovis ariea ariea), cattle
(Bos primigenius taurus), goat (Capra hircus), and their wild relatives
(deer and muflon, etc.) have an important role in the nutrient chain and
in the economy. Their special importance is that they live on feedstuffs
which cannot be utilized by other herbivores. The forestomachs provide
anaerobic environment for the rumen flora, which is able to digest
cellulose and utilizes non-protein nitrogen beside the common nutrients.
The composition of rumen flora largely depends on the fed ration and
adapts to the diet. This adaption, however, takes several days and the
final stabilization may require several weeks. Abrupt change of the ration
adversely affects intake, digestibility and production, and it may cause
illness or even death.
It is well known that in 1 ml of rumen liquor millions of bacteria live and
grow. Anaerobic fermentation by bacteria is of high importance for the
normal digestion and feed intake. Energy producing nutrients are fermented
to acetic, propionic and butyric acids, which are used by the host animal
as fatty acids; the bacterial mass passing to the intestines will be
digested and used as protein source. With respect to the milk and meat
production the supply of acetic acid and propionic acid and their mutual
proportion play an essential role. Therefore, the rumen flora has an
important role in the maintenance and production of the ruminant farm
animals.
After birth the rumen flora develops spontaneously and attains its adult
composition after weaning to solid food. It is not sure, however, that
this accidental flora would represent the optimum fermentative system. It
would be advantageous to have a process for the modulation of the
composition and/or of the number of the rumen flora according to economic
interests.
The increase of volatile fatty acid production in the rumen and thereby the
improvement of feed utilization and, accordingly, meat or milk production
is a long felt want in the animal husbandry. Certain results have been
obtained with monensin
[2-[5-ethyltetrahydro-5-{tetrahydro-3-methyl-5-[tetrahydro-6-hydroxy-6-(hy
droxymethyl)-3,5-dimethyl-2H-pyran-2-yl]-2-furyl}-2-furyl]-9-hydroxy-.beta.
-methoxy-.alpha.,.gamma.,2,8-tetramethyl-1,6-dioxaspiro[4.5]decane-7-butyri
c acid] originally used as coccidiostat (see e.g. the U.S. Pat. No.
4,085,255). Experimental use of other related polyethers, such as
salinomycin, lasalocid, etc., phthalide derivatives (U.S. Pat. No.
4,333,923) and glycopeptides, such as avoparcin, actaplanin and the like
(Ingle et al., Abstr. Am. Soc. Anim. Sci. 424/1978/) has also been
reported. The effect observed is, however, strongly different on animals
fed on various feedstuffs and is not substantial altogether (Chalupa, W.,
Chemical Control of Rumen Microbial Metabolism, Digestive Physiology and
Metabolism in Ruminants, MTP Press, Lancaster, England, 1980 and Chalupa,
W. et al., Manipulating Rumen Fermentation with Monensin and Amicloral,
Abstr. Am. Soc. Anim. Sci., 410/1978/). There is no method known in the
art by which directly the microbial culture present in the rumen of
ruminants could be influenced to provide significant results.
During our experiments we have found that genetic recombination methods can
be successfully used for the separation of microorganisms. Practically any
bacterial strain can be labelled by genetic markers, e.g. by an antibiotic
resistance factor that allows identification of the bacterium among other
bacteria.
We isolated rumen bacteria, genetically labelled the strains and after
culturing we reintroduced them into the rumen. Then we took samples of
rumen content periodically, cultured them in selective media, and found
that some of the strains, that had fermentative characteristics
advantageous for the host animal and that were able to grow in vitro,
could grow in the rumen and persist there for a long time, if the same
feed-stuff was fed as during isolation, and they stimulated digestion and
thereby feed utilization by the host animal.
The instant invention relates to a composition which comprises as active
ingredient one or more microbial cultures, capable of adjusting the weight
ratio of acetic acid to propionic acid to an optimum value of 1.5-4.0:1,
and of growing in the rumen and persisting there at least 60 days,
optionally in admixture with carriers, diluents, preserving agents
conventionally used in animal husbandry and nutrition and/or other
substances conventionally administered to ruminants.
If compositions are to be used to increase meat production, the active
ingredient preferably is a microbial culture capable of adjusting the
ratio of acetic acid to propionic acid to 2.0-3.5:1, e.g. 2:1. For milk
production the optimum acetic acid to propionic acid ratio is about 3.0:1,
for subsistence or gestation about 4.0:1, and for heifer breeding about
2.0-3.0:1. It is, therefore, advisable to use compositions capable of
adjusting the acetic acid to propionic acid ratio to the optimum value for
these purposes. In the literature there is some uncertainty as to the most
desired acetic acid to propionic acid ratios, the preferred ratio being a
function of the ruminant, the feedstuff employed and other factors and its
selection is the task of those skilled in the art (see e.g. Kaufmann, W.
and Rohr, K., Der Einfluss des Futters auf die bakterielle Fermentation in
Vormagen, Handbuch der Tiernahrung, 263, 1969, Parey, Hamburg, Berlin).
The invention further relates to a process for the preparation of microbial
cultures used as active ingredient in the above compositions, in which
samples are taken from the rumen of animals fed on a given feedstuff or
ration,
metabolism of microbes isolated from the sample is examined in vitro, and
microbes with advantageous metabolic characteristics are cultivated in
media containing the same feedstuff or ration as carbon- or
nitrogen-source,
a genetic marker, which makes selection possible, is introduced into the
growing microbes,
the genetically labelled strains are cultivated,
the cultures are reintroduced into the rumen of the animals fed on the same
feedstuff or ration,
samples are taken from the rumen,
the cell number of the genetically labelled strain is counted,
strains persisting for at least 60 days and adjusting the acetic acid to
propionic acid ratio to an optimum value, preferably to 1.5-4.0:1, are
separated, and
if desired, these strains are formulated i a form acceptable for the
practice of animal husbandry and nutrition.
According to a preferred embodiment of the process of the invention samples
are taken from the rumen of a fistulated ruminant fed on hay, cereal meal
or molasses, and the cultures containing rumen bacteria are spread on
solid media containing N- and C-sources, inorganic salts, rumen liquor and
agar (Bryant and Burkey, J. Dairy Sci., 36, 206, 1953). The cultures are
incubated in anaerobic conditions, then the clones are isolated and
cultured in similar media as above.
The cultures grown out are inoculated into liquid media containing
nitrogen-source, inorganic salts, rumen liquor, and as a carbon-source,
hay or cellulose, or in other cases cereal meal or molasses, and they are
incubated until intensive growth begins in the media.
Cells grown on hay (cellulose), cereal meal (starch) or molasses (sucrose)
as carbon sources are spread, cultured and isolated on solid media
containing cellulose (for cells grown with hay), glucose (for cells grown
with cereal meal) or sucrose (for cells grown with molasses) as carbon
source.
The cultures obtained are labeled genetically. For labeling any inheritable
genetic marker can be used that allows the identification of the labeled
microbe among other microbes.
According to a further preferred embodiment of the invention, antibiotic
resistance genes are introduced into the selected cells. If the bacteria
living in the rumen are sensitive to a certain antibiotic and resistance
cells are mixed to them, growth and death of the latter microbes may be
followed easily if the samples are spread on media containing the same
antibiotic. In this case only the resistant cells will grow.
By transformation (Bergmans et al., J. Bacteriol. 146, 564/1981/) we
introduce p1011 plasmid (Simond et al., Proc. 8th North American Rhizobium
Conference, Winnipeg, Canada, Univ. of Manitoba Press, 1983) carrying
kanamycin and chloramphenicol resistance genes into the selected cultures
which grow on cellulose, cereal meal or molasses. Plasmid DNA was isolated
from E. coli cells (Birnboim and Doly, Nucl. Acid. Res. 7, 1513/1979/).
The strain is deposited in the National Collection of Agricultural and
Industrial Microorganisms, Budapest, Hungary, under Accession No. NCAIM
B(P) 00264.
After transformation with DNA carrying antibiotic resistance genes, cells
containing and expressing the new genetic information are selected on
solid media with the above-mentioned composition, but supplemented with
kanamycin. The resistant strains will be stored.
With the isolated and stored cultures media containing nitrogen-source,
inorganic salts, rumen liquor and agar (to achieve semi-solid consistency)
are inoculated and incubated under anaerobic conditions. The developed
cultures are mixed to the feed of sheep starved for one day. Before and
after feeding bacteria to the animal, rumen samples are taken daily, the
samples are spread on the above-described solid media containing
kanamycin, and bacterial cells resistant and sensitive to the antibiotic
are counted. Ruminal production of volatile fatty acids is also determined
qualitatively and quantitatively. It is known that feed utilization by
ruminants is affected by the ratio of volatile fatty acids (Eskeland et
al., J. Anim. Sci. 33, 282/1971/; Church et al., Digestive Physiology and
Nutrition of Ruminants, Vol. 2, pp. 622-625, 1971/. As mentioned above,
the optimum ratio of acetic acid to propionic acid is considered to be
2.0-3.5:1, 3:1, and 4:1, respectively, for growing, milk production, and
maintenance as well as pregnancy, resp. (Kaufmann, W. and Rohr, K.: Der
Einfluss des Futters auf die bakterielle Fermentation in Vormagen. In:
Hanbuch det Tierernahrung, p. 263 , Parey, Hamburg-Berlin, 1969).
According to a preferred embodiment of the process of the invention
bacteria are isolated from rumen samples and the capacity of the isolates
to produce volatile fatty acids is examined. The microorganism is cultured
in anaerobic conditions in the described complete media containing rumen
liquor, then acetic, propionic and butyric acid concentrations of the
cultures are determined. Microbial cells producing volatile fatty acids in
required ratios are labelled genetically and their ruminal growth are
examined.
Strains, that are able to grow in the rumen of the animal fed on the
described feedstuff at least for 60 days and that can ferment dietary
carbohydrates to volatile fatty acids in optimum ratios, are selected,
grown, isolated, maintained and stored, and, if desired, their cultures,
in a form acceptable for animal husbandry, will be orally administered to
ruminants, for developing or modulating the rumen flora.
Three strains, Hh-GYOKI-1-123Sz, Hh-GYOKI-2-14Ab and Hh-GYOKI-3-81Me,
capable of growing in the rumen of animals fed on hay, cereal meal or
molasses, respectively, that could persist in the rumen for a long and
affected digestion advantageously, have been deposited in the National
Collection of Agricultural and Industrial Microorganisms, Budapest,
Hungary, under Accession Nos. NCAIM B(P) 000287, NCAIM B(P) 000288 and
NCAIM B(P) 00298 respectively.
The microorganism Hh-GYOKI-1-123 Sz, which was isolated from rumen,
biosynthesizes propionic acid. The bacterium is of Gram-positive coloring
and rodlet-shaped. It ferments glucose and starch to propionic acid and
acetic acid while some butyric acid and carbon dioxide respectively, are
being formed. The cells which are rodlet-shaped in their early age on
complete culture media will change later to pleomorphous form without
cilium, often widening on their tips and having an irregular form and
size. It readily grows under anaerobic and semi-anaerobic conditions, too,
it is facultatively anaerobic.
On the above basis the strain Hh-GYOKI-1-123 Sz, can be assigned to the
Propionibacterium genus of the Propionibacteriaceae family. (The
microorganism Hh-GYOKI-48a originating from Hh-GYOKI-1-123 can be
similarly assigned to the Propionibacterium genus.)
The microorganism Hh-GYOKI-2-14 Ab isolated from rumen produces propionic
and acetic acids, it is unmoving and of spherical form, it is of
Gram-negative coloring. It readily grows also under anaerobic conditions
at a temperature of 37.degree. C. The cells of spherical form are settled
down in great quantities beside each other, their average size is of
0.3-0.4 .mu.. It ferments acid and carbon dioxide from carbohydrates and
produces hydrogen sulfide on complete culture media. It does not liquefy
gelatine, and does not haemolyze. On the basis of the above features the
microorganisms Hh-GYOKI-2-14 Ab can be assigned to the slightly known
Veillonella genus.
The microorganisms Hh-GYOKI-3-81 Me produces acetic acid and is
facultatively anaerobic. The cells do not move, are rodlet-shaped, and
their ends are flattened. They are of Gram-positive coloring. In addition
to acetic acid they produce lactic acid from glucose. On different culture
media the rodlets rarely stand singly, more often in a chain. Producing of
pigments cannot be observed.
Based on the above facts, the microorganisms Hh-GYOKI-3-81 Me can be
assigned within the family Lactobacteriaceae to the genus Bifidobacteria.
The determinations of species have been made according to the following
literature; Bergey's Manual of Determinative Bacteriology; Breed, Murray
and Hitchens. The Williams and Wilkins Company, Baltimore, 1948, 8th
edition.
According to the invention it is preferred to culture the microorganisms of
rumen origin between 32.degree. C. and 37.degree. C., under anaerobic
conditions, with the exclusion of oxygen, in media containing carbon- and
nitrogen-sources, inorganic salts, reducing agents and rumen liquor; the
latter provides growth factors. As carbon source glucose, cellulose, hay,
cereal meal or molasses can be used, while inorganic salts, yeast extract,
casein and similar additives are suitable N-sources.
An essential feature of the invention is that unicellular organisms
advantageously fermenting the fed feedstuff or ration and capable of
persisting in the rumen for a long period are used for the modulation of
ruminal flora. For the selection of such strains genetic markers are used,
as described above, e.g., genes coding antibiotic resistance, enzyme
proteins or other detectable proteins. Auxotrophic cells can be used, too.
The genetic marker is introduced into the cell by a vector DNA molecule,
e.g. by a plasmid or phage, but selectable characteristics, e.g.
resistance to an antibiotic, can be chosen by spontaneous selection, too.
The selected strains, which have advantageous fermentative characteristics
and persist in the rumen for a long period, are used either to enhance the
development of rumen flora in suckling ruminants or to modify
advantageously the composition of the established rumen flora. It is
recommended to administer the preparation with the feed or drinking water.
The microorganism selected for making the preparation according to the
invention is cultured in media containing organic carbon source, organic
or inorganic nitrogen source and organic and inorganic salts, and is then
isolated in a form suitable for oral administration or for transport. If
desired, the microorganism culture is formulated by mixing it with solid
or liquid carriers or other additives. The preparation can be mixed to the
feed or drinking water, or can be fed alone.
In case of sheep, for example 1 to 20 g, preferably 5 g, of microorganism
culture according to the invention are added to about 0.5 kg of feed. As a
feedstuff e.g. a mixture of corn-meal, lucerne hay and beef cattle feed
can be used, and the actual proportions should be determined in view of
the actual conditions and the desired daily gain in weight. Cattle are
generally administered 10 to 200 g, preferably 50 g, of a microorganism
culture according to the invention per day, e.g. in admixture with about 5
kg of a conventional feedstuff.
The method of improving the efficiency of feed utilization of ruminants is
also within the scope of the invention.
After culturing in liquid media, as mentioned hereinbefore, the
microorganisms are separated by centrifugation or filtration. Pastes,
freeze dried preparations or suspensions containing spores or vegetative
forms, etc. may be prepared and additives acceptable for animal husbandry
and nutrition may be added. Other additives, e.g. proteins, amino acids or
glycerol, may help to keep the microorganisms viable. To the compositions
according to the invention used to improve feed utilization in ruminants
other substances conventionally used in the practice may also be added,
e.g. antibiotics that stimulate the growth of the host animal (monensin,
nigericin, salynomycin etc.) or enhance the persistency of the
microorganisms fed.
So the microbial cultures according to the invention enable the formation
of a living microbial culture in the rumen or the advantageous
modification of an established flora.
During the suckling period the rumen flora is unable to effectively ferment
the common feedstuffs. The flora develops spontaneously and accidentally,
and it is by no means certain that its composition is optimal for the host
animal.
By feeding the selected microbial strains, instead the slow and spontaneous
development of the rumen flora, a rapid development may be achieved, and
the rumen flora will be capable of optimally utilizing the feed.
The advantage of the instant process is that with the microorganisms
prepared in the described way (e.g. with the strains NCAIM B(P) 000287,
NCAIM B(P) 000288 and NCAIM B(P) 000289) we can promote the rapid adaption
or development of rumen flora during feed change or weaning, by enhancing
ruminal growth of microorganisms capable of optimal degradation of the
feed.
The process can be used, among others, in the following cases:
for dairy cows during changes of lactation, at the end of pregnancy and
during seasonal and other changes of the ration;
for beef cattle at the beginning and end of the grazing period, at the
change of fattening with roughages to an intensive fattening with cereal
meal, and during other changes of the growing-fattening diet;
for sheep during the seasonal changes of feeding, at the beginning and end
of the grazing period and during the commencement of an intensive growing
and fattening.
The possibilities are similar in the goat husbandry, too. Microbial
cultures prepared by the process of the invention may be used also in
several special cases, e.g. for wild-living ruminants, in game preserves
and for the fallow-deer.
It should be noted that although the microbial cultures used in the
compositions according to the invention preferably are of rumen origin,
other acetic acid and/or propionic acid producing bacteria, which do not
necessarily originate from the rumen, are also suitable. Such bacteria
include certain members of the genus Angerovibrio (lipolytical),
Bacteroides,
Selenomonas (ruminanticum) and Propionibacteria.
The invention will further be illustrated by the aid of the following,
non-limiting Examples. The preparation of microbial strains which are
capable of utilizing basic rations containing mainly cellulose (hay),
starch (cereal meal) or sucrose (molasses) and persist in the rumen for a
long period will be described in detail. The use of the preparation is
described for sheep, but the scope of protection extends to microorganisms
capable of growing on other feedstuffs and to the development or
modification of the rumen flora of other ruminant species as well.
EXAMPLE 1
Modification of the rumen flora of animals fed on hay.
A) Isolation of microorganisms able to grow on hay
Sheep are laparatomized fitted with rumen fistula and fed on hay for a
month. Rumen sample is taken through the fistula, diluted and spread on
RGCA solid media of following composition:
______________________________________
Salt solution I:
K.sub.2 HPO.sub.4 0.6 g
distilled water ad 100.0 g
Salt solution II:
NaCl 1.2 g
(NH.sub.4).sub.2 SO.sub.4
1.2 g
KH.sub.2 PO.sub.4 0.6 g
CaCl.sub.2 0.12 g
MgSO.sub.4.7H.sub.2 O 0.25 g
distilled water ad 100.0 ml
Resazurin (0.1% solution)
0.1 ml
Agar (Bacto).sup.x 2.5 g
Rumen liquor.sup.xx 10.0 ml
Glucose 0.05 g
Cellobiose 0.05 g
Cystein.HCl monohydrate 0.05 g
Sodium carbonate (8% solution)
5.0 g
Distilled water ad 50.0 ml
______________________________________
.sup.x Difco Labs, Detroit, USA
.sup.xx The sample of rumen content is filtered through several layers of
gauze, then the filtrate is stored under carbon dioxide at -20.degree. C.
Before sterilization under CO.sub.2 gas, the pH of the media RGCA is
adjusted to 6.8. Sterilization, preparation of the media and cultivation
are performed according to Bryant and Burkey (J. Dairy Sci., 36,
205/1953/).
The rumen liquor is diluted with a sterile mixture of the following
composition:
______________________________________
salt solution I (see above)
7.5 ml
salt solution II (see above)
7.5 ml
cystein.HCl monohydrate 0.05 g
Na.sub.2 CO.sub.3 0.3 g
resazurin (0.1% solution)
0.1 ml
distilled water ad 100.0 ml
______________________________________
The name of this mixture is HB.
The cultures are incubated in anaerobic conditions at 35.degree. C. (see
Atlas of Rumen Microbiology, Ogimoto and Imai, Japan Scientific Societies
Press, Tokyo, 1981) for 120 hours, then the individual clones are
inoculated into media, containing extracted hay, of the following
composition:
______________________________________
salt solution I (see above)
15.0%
salt solution II (see above)
15.0%
resazurin (0.1% solution)
0.1%
Tripton L42 (Oxoid).sup.x
15.%
yeast extract (Oxoid).sup.x
0.5%
rumen liquor.sup.xx 10.0%
Na.sub.2 CO.sub.3 0.4%
cystein.HCl monohydrate
0.05%
extracted hay.sup.xxx
10.0%
______________________________________
Name of the media: RGCF liquid media
.sup.x Oxoid Ltd., London, UK.
.sup.xx See above
.sup.xxx For preparing extracted hay finely cut hay particles are
suspended in water, boiled and filtered. The filtration rest is added to
the media before sterilization.
Before sterilization the pH of the media is adjusted to 6.5.
Test tubes containing 5 ml of sterile media are inoculated with the
microbial suspension obtained from individual clones grown on RGCA solid
media and incubated under anaerobic conditions at 35.degree. C. The growth
is checked by microscopic examination and the cultures are spread on RGCA
solid media where 2.0% Bacto cellulose are substituted for the glucose and
the cellobiose.
The cultures are incubated in anaerobic conditions at 35.degree. C. for 120
h, then individual clones made up of cells utilizing cellulose are
inoculated onto RGCA media containing cellulose.
This way ruminal microorganisms able to grow on hay or cellulose can be
obtained.
B) Genetic labelling of rumen bacteria able to grow on hay
Genetic labelling is performed with E. coli p1011 plasmid, according to
Simon et al. (Proc. 8th North American Thizobium Conference, Winnipeg,
Canada, Univ. of Manitoba Press, 1983).
The plasmid DNA is isolated from an E. coli culture according to Birnboim
and Doly (Nucl. Acid Res. 7, 1513/1979/) and is dissolved in an aqueous
solution containing the following components:
______________________________________
75 mM CaCl.sub.2
5 mM MgCl.sub.2
10 mM tris.HCl buffer.sup.x, pH 7.5
______________________________________
.sup.x tris(Hydroxymethyl)-aminomethane hydrochloride
Microbes able to grow on hay and isolated according to item A) of Example 1
are cultured on RGCF media and separated by centrifugation under CO.sub.2.
The cells are suspended in an aqueous solution containing in 1 liter the
following components:
75 mM CaCl.sub.2
5 mM MgCl.sub.2
10 mM tris.HCl buffer, pH 7.5
1 nM crystein.HCl monohydrate
1 mM sodium thiosulfate
wherein the suspensions should contain 5.times.10.sup.9 cells per ml. The
suspension is diluted with the same volume of solution containing plasmid
DNA (0.1 .mu.g/ml) and is incubated for 60 min. at 4.degree. C. Then the
incubation is continued at 41.degree. C. for 2 min., then the culture is
spread on solid media containing 500 .mu.g/ml of kanamycin B, and
cellulose as a carbon source. The culture is incubated at 35.degree. C.
for 120 h under anaerobic conditions and clones able to grow in the
presence of 500 .mu.g/ml of kanamycin B are examined.
Plasmid p1011 carries genes determining resistance to kanamycin and
chloramphenicol; furthermore, it contains replication origin allowing the
start of replication in E. coli cells. If transformed into other bacteria,
owing to its lack of suitable origin allowing replication, the plasmid DNA
is either eliminated or incorporated into the chromosome (partly or
completely) and genetic recombination takes place. Eventually the gene
incorporated into the chromosome is expressed and endows the cells with
kanamycin and chloramphenicol resistance.
In our experiments we obtained kanamycin B resistance clones with
transformational frequency of 3.times.10.sup.-5.
Several resistant clones were isolated and we determined the sensitivity to
antibiotic of the initial and kanamycin B resistant (Km.RTM.) strains.
Results obtained with several strains are shown in Table 1.
TABLE 1
______________________________________
Sensitivity to kanamycin B of rumen bacteria and
of strains labelled genetically and degrading hay
Least effective concentration
Microbe of kanamycin B, .mu.g/ml
______________________________________
Rumen liquor 31
Initial strains 4.0 to 7.5
Genetically labelled Km .RTM. strains
Hh-GYOKI-1-8 500
27 250
91 500
123 1000
142 500
______________________________________
So we can obtain microorganisms of rumen origin that are able to utilize
hay or cellulose and to grow in the presence of high amounts of kanamycin
B.
The strain Hh-GYOKI-1-123 (Km.RTM.) is spread on RGCA media containing
cellulose, whereafter kanamycin B in concentrations of 1000, 5000 and
10,000 .mu.g/ml are added. The cultures are incubated in anaerobic
conditions at 35.degree. C. for 168 h and the strains growing in presence
of 10,000 .mu.g/ml antibiotic are isolated. So, with spontaneous
selection, we obtain spontaneous mutants highly resistant to kanamycin B.
One of these strains has been designated Hh-GYOKI-1-123Sz and deposited in
the Hungarian National Collection of Medical Bacteria of the National
Institute of Hygiene, Budapest, under No. 00287.
C) Reintroduction of labelled microbes into the rumen
Sterile, solid media named RGCFa are inoculated with the culture of strain
Hh-GYOKI-1-123 (Km.RTM.) stored on RGCA slants at +4.degree. C. The
composition of RGCFa media is as follows:
______________________________________
1. K.sub.2 HPO.sub.4
0.3% 45 ml solution
2. (NH.sub.4).sub.2 SO.sub.4
0.6% 45 ml solution of the mixture
NaCl 0.6%
MgSO.sub.4.2H.sub.2 O
0.06%
CaCl.sub.2.2H.sub.2 O
0.06%
KH.sub.2 PO.sub.4
0.3%
3. Cellulose (Bacto).sup.x
1.8% 65 ml solution of the mixture
Agar (Bacto).sup.x
3.0%
4. Yeast extract 0.1% 20 ml solution
5. Cystein.HCl.H.sub.2 O
0.1% 20 ml solution
6. Sodium thiosulfate
0.1% 10 ml solution of the mixture
Na.sub.2 CO.sub.3
0.2%
7. Rumen liquor.sup.xx 20 ml
______________________________________
.sup.x Difco Labs, Detroit, USA
.sup.xx See above
The 7 solutions are prepared separately and mixed in the given sequence.
Cultivation is performed in 500 ml Erlenmeyer flasks containing 150 ml of
media, under anaerobic conditions. Growth is checked after 48 hours, then
the culture is mixed to the feed of a hay-fed sheep. Three hundred forty
ml of culture containing 4.7.times.10.sup.6 bacteria per ml were orally
administered to the sheep. Before administration and on the consecutive
days 50 to 200 ml samples are taken through the rumen fistula. The samples
are diluted with HB solution and spread on RGCA media lacking kanamycin B
or other antibiotics. The cultures are incubated in anaerobic conditions
at 35.degree. C. for 72 hours and the bacterial clones are counted.
Results are shown in Table 2.
TABLE 2
______________________________________
Changes of rumen flora of a sheep treated
with strain Hh-GYOKI-1-123 (Km .RTM.)
Cell count/ml
Without In the presence of 1000 .mu.g/ml
Sample antibiotics
of kanamycin B
______________________________________
Before 5 .times. 10.sup.6
0
administration
After
administration
day 1 5.9 .times. 10.sup.6
2.0 .times. 10.sup.4
day 2 3.2 .times. 10.sup.7
4.1 .times. 10.sup.4
day 3 2.4 .times. 10.sup.6
3.1 .times. 10.sup.4
day 6 3.9 .times. 10.sup.7
1.8 .times. 10.sup.4
day 8 9.8 .times. 10.sup.6
1.05 .times. 10.sup.5
day 15 8.1 .times. 10.sup.5
3.1 .times. 10.sup.4
______________________________________
It is seen from Table 2 that the microorganism administered to the animal
persists and grows in the rumen.
According to the above-mentioned process we also cultivated the strain
Hh-GYOKI-1-123Sz resistant to 10,000 .mu.g/ml kanamycin B, and orally
administered it to the same sheep on the 15th day.
Hundred forty ml culture containing 2.times.10.sup.8 bacterial per ml were
administered orally.
Bacteria of rumen samples were cultivated and counted as before. Results
are shown in Table 3.
TABLE 3
______________________________________
Changes in the rumen flora of a sheep treated
with strain Hh-GYOKI-1-123Sz (Km .RTM.)
Cell count/ml
Without In the presence of 8000 .mu.g/ml
Sample antibiotics
of kanamycin B
______________________________________
Before 1.4 .times. 10.sup.6
0
administration
After
administration
day 1 7.4 .times. 10.sup.6
3.2 .times. 10.sup.4
day 2 1.7 .times. 10.sup.5
1.3 .times. 10.sup.4
day 5 4.0 .times. 10.sup.6
9.1 .times. 10.sup.3
day 7 1.1 .times. 10.sup.7
2.0 .times. 10.sup.4
day 14 8.0 .times. 10.sup.6
3.1 .times. 10.sup.4
day 21 7.1 .times. 10.sup.5
6.2 .times. 10.sup.4
day 28 8.2 .times. 10.sup.6
8.1 .times. 10.sup.4
day 35 8.7 .times. 10.sup.6
1.8 .times. 10.sup.4
______________________________________
The data of Table 3 indicate that the microorganism administered is present
in significant quantities in the rumen and, because the fluid phase of the
rumen content is continuously emptied, it surely replicates. Differences
of bacterium counts between samples may be explained by variations of the
consistency of rumen content from thick to fluid.
EXAMPLE 2
Modification of rumen flora in a sheep fed on cereal meal
A) Isolation of microorganisms able to grow on cereal meal
The process described under item A) of Example 1 is repeated with the
difference that the initial sample is taken from the rumen of a sheep fed
on cereal meal, the individual isolates are inoculated into RGCF liquid
media (see above) containing 2% of cereal meal powderized in a mortar,
instead of extracted hay, cultures grown in RGCF liquid media are spread
on RGCA solid media (see above) and clones developed from cells able to
utilize cereal meal are isolated on similar media.
In this way microorganisms able to grow on cereal meal as a carbon source
are obtained.
B) Genetic labelling of rumen bacteria utilizing cereal meal
The process described under item B) of Example 1 is repeated with the
difference that strains obtained according to item A) of Example 2 are
used for transformation by p1011 plasmid DNA, instead of those obtained
according to item A) of Example 2.
Resistance to kanamycin B of several transformed and Km.RTM. strains is
shown in Table 4.
TABLE 4
______________________________________
Sensitivity to kanamycin B of rumen bacteria and of -strains labelled
genetically and utilizing cereal meal
Lowest kanamycin
B concentration
Microorganisms inhibiting growth, .mu.g/ml
______________________________________
Rumen liquor 31
Initial strains 1.8
Genetically labelled Km .RTM. strains
Hh-GYOKI-2-4 250
14Ab 250
37 250
81 125
______________________________________
By performing the above-mentioned process microbes of rumen origin are
obtained that are able to utilize cereal meal as carbon source and are
eight times more resistant to kanamycin B than the rumen flora.
The strain designated as Hh-GYOKI-2-14Ab has been deposited in the National
Collection of Agriculture and Industrial Microorganisms, Budapest,
Hungary, under Accession No. NCAIM B(P) 000288.
C) Reintroduction of microorganisms labelled genetically into the rumen
The process described under item C) of Example 1 is repeated with the
difference that sterile RGCFa media containing 1.8% of starch, instead of
1.8% of cellulose (Bacto), is inoculated, mixed into the feed of the sheep
and samples are taken daily through the fistula before administration and
after administration. Three hundred ten ml culture containing
7.1.times.10.sup.5 bacteria/ml were administered orally to the sheep.
TABLE 5
______________________________________
Changes in the rumen flora of the a sheep treated
with strain Hh-GYOKI-2-14Ab (Km .RTM.)
Cell number/ml
Without In the presence of 250 .mu.g/ml of
Sample antibiotics
kanamycin B
______________________________________
Before 4.6 .times. 10.sup.6
1.4 .times. 10.sup.1
administration
After
administration
day 1 4.1 .times. 10.sup.6
1.8 .times. 10.sup.3
day 2 3.2 .times. 10.sup.6
2.1 .times. 10.sup.3
day 3 8.0 .times. 10.sup.3
1.1 .times. 10.sup.3
day 6 9.1 .times. 10.sup.6
7.1 .times. 10.sup.2
day 8 8.0 .times. 10.sup.6
8.1 .times. 10.sup.3
day 15 6.8 .times. 10.sup.6
8.7 .times. 10.sup.3
day 22 5.0 .times. 10.sup.6
9.8 .times. 10.sup.3
day 29 8.0 .times. 10.sup.6
1.1 .times. 10.sup.4
day 36 9.1 .times. 10.sup.6
7.0 .times. 10.sup.3
day 43 7.0 .times. 10.sup.6
7.9 .times. 10.sup.3
day 60 6.1 .times. 10.sup.6
7.0 .times. 10.sup.3
______________________________________
The data of Table 5 indicate that the microorganism administered persists
and replicates in the rumen for a long time.
EXAMPLE 3
Modification of the rumen flora of a sheep fed on molasses
A) Isolation of microorganisms able to grow on molasses
The process described under item A) of Example 1 is repeated with the
difference that the initial samples are taken from a sheep fed on
molasses, the individual isolates are inoculated to RGCF media containing
glucose instead of extracted hay, the cultures grown in liquid media are
spread on RGCA media and clones grown from cells utilizing molasses are
inoculated on the same RGCA media.
So microorganisms of rumen origin utilizing molasses are obtained.
B) Genetic labelling of bacteria utilizing molasses
The process described under item B) of Example 1 is repeated with the
difference that cells prepared according to item A) of Example 3 are
transformed by p1011 plasmid DNA, instead of those obtained according to
item A) of Example 1.
The resistance to kanamycin B of several Km.RTM. strains is shown in Table
6.
TABLE 6
______________________________________
Resistance to kanamycin B of rumen bacteria and
of genetically labelled strains utilizing molasses
Lowest concentration of
Microorganisms
kanamycin B inhibiting growth
______________________________________
Rumen liquor 31
Initial strains
7.5
Genetically labelled
Km .RTM. strains
Hh-GYOKI-3-2 250
14 500
34 250
81Me 500
132 500
______________________________________
In this way isolates highly resistant to kanamycin B and utilizing molasses
are obtained.
The strain signed Hh-GYOKI-3-81Me has been deposited in the Hungarian
National Collection of Medical Bacteria of the National Institute of
Hygiene, Budapest under No. 00289.
C) Reintroduction of genetically labelled bacteria into the rumen
The process described under item C) of Example 1 is repeated with the
difference that the strain Hh-GYOKI-3-81Me is inoculated onto RGCFa media
containing 1.8% of glucose instead of 1.8% of cellulose, and the culture
is mixed with the feed of a sheep fed on molasses. Three hundred eighty ml
of a culture containing 1.6.times.10.sup.7 bacterial per ml were orally
administered. One sample each will be taken daily before and after
administration through a rumen fistula.
TABLE 7
______________________________________
Changes in the rumen flora of a sheep treated
with strain Hh-GYOKI-3-81Me (Km .RTM.)
Cell number/ml
Without In the presence of 500 .mu.g/ml of
Sample antibiotics
kanamycin B
______________________________________
Before 5.0 .times. 10.sup.6
0
administration
After
administration
day 1 1.1 .times. 10.sup.6
1.9 .times. 10.sup.5
day 2 1.8 .times. 10.sup.7
2.5 .times. 10.sup.5
day 3 6.2 .times. 10.sup.6
6.1 .times. 10.sup.5
day 6 8.1 .times. 10.sup.6
8.7 .times. 10.sup.5
day 8 6.2 .times. 10.sup.6
9.1 .times. 10.sup.5
day 15.sup.x
1.3 .times. 10.sup.3
1.3 .times. 10.sup.2
day 22 3.0 .times. 10.sup.6
3.1 .times. 10.sup.5
day 29 1.1 .times. 10.sup.6
7.0 .times. 10.sup.5
day 36 8.0 .times. 10.sup.5
9.1 .times. 10.sup.4
day 43 6.1 .times. 10.sup.6
8.1 .times. 10.sup.5
day 60 6.8 .times. 10.sup.6
6.4 .times. 10.sup.5
______________________________________
.sup.x Sampling error
The data indicate that the microorganism administered persists for a long
period in the rumen of sheep fed on molasses.
EXAMPLE 4
Changes in ratios of volatile fatty acids owing to treatment with the
bacterial preparation
Two sheep are fed on a complete ration for 14 days, then rumen sample is
taken through a fistula. Two liters of rumen liquor are filtered through
several layers of gauze. The particulate rest is suspended in 1 liter of
physiological buffer (see below), mixed and filtered as before. The two
filtrates are mixed, left to stand for an hour, solids floating on the
surface are discarded and the liquid phase is used for the examination.
The composition of the physiological buffer is as follows:
______________________________________
Na.sub.2 HPO.sub.4
0.316 g/l
KH.sub.2 PO.sub.4
0.152 g/l
NaHCO.sub.3 2.260 g/l
KCl 0.375 g/l
NaCl 0.375 g/l
MgSO.sub.4 0.112 g/l
CaCl.sub.2.H.sub.2 O
0.050 g/l
FeSO.sub.4.7H.sub.2 O
0.008 g/l
MnSO.sub.4.H.sub.2 O
0.004 g/l
ZnSO.sub.4.7H.sub.2 O
0.004 g/l
CuSO.sub.4.5H.sub.2 O
0.002 g/l
CoCl.sub.2.6H.sub.2 O
0.001 g/l
______________________________________
The pH of the mixture is checked and, if required, adjusted to pH 7.2 with
an aqueous HCl or NaCH solution (Cheng et al.: J. Dairy Sci. 38,
1225/1955/).
To the mixture obtained the same volume of physiological buffer is added
and in 1 liter of the diluted mixture 4 g of the ration is suspended.
Thirty ml each of the suspension is poured into Erlenmeyer flasks of 100
ml volume. 200 doses are sterilized and another 200 are not.
Sterile media and media containing living rumen bacteria are inoculated
with bacterial strains to be examined for producing acetic, propionic and
butyric acids.
Bacterial strains proven to be able to persist in the rumen for a long time
after an in vitro cultivation will be examined. In addition, bacterial
strains isolated from the rumen liquor of a sheep fed on complete or any
ration according to items A, B and C of Example 1 are examined, too.
Bacteria to be examined are cultivated on RGC+CG media (see below) in
anaerobic conditions at 37.degree. C. for 48 hours.
______________________________________
Composition of RGC + CG media:
______________________________________
salt solution I (see item A of Example 1)
15%
salt solution II (see item A of Example 1)
15%
trace element solution.sup.x
0.3%
yeast extract (Oxoid) 0.5%
filtered rumen liquor 10.0%
Na.sub.2 CO.sub.3 0.4%
cystein.HCl.H.sub.2 O 0.05%
sodium thiosulfate 0.008%
cellulose (Bacto) 0.3%
glucose 2.0%
______________________________________
.sup.x Composition of the trace element solution:
ZnCl.sub.2 40 mg
CuCl.sub.2.2H.sub.2 O
10 mg
disodium tetraborate dekahydrate
10 mg
ammonium molybdenate tetrahydrate
10 mg
FeCl.sub.3.6H.sub.2 O
200 mg
MnCl.sub.2.4H.sub.2 O
10 mg
deionized water ad 1000 ml
Cultures are inoculated into media prepared in Erlenmeyer flasks. Two ml of
culture each is inoculated into 50 ml of media, in two parallel flasks.
Non-sterile cultures are inoculated, too.
The flasks are incubated under anaerobic conditions for 40 hours. The
growth is stopped with 10% formic acid solution and the volatile fatty
acid content of the cultures is examined.
The cultures are filtered through gaze layers and centrifuged at 4000 rev.
per min. for 15 min., then filtered again and brought onto the separation
column of a Carlo Erba GI-452 gas-liquid chromatograph, fitted with flame
ionization detector, for determining the C.sub.2 -C.sub.5 fatty acids.
Temperature of the column: 150.degree. C.
Separation column: 2 m long, 4 mm wide (inner diameter) glass tube filled
with 10% of ethylene glycol adipate and 2% of o-phosphoric acid on a
silanated silica gel carrier (0.2 to 0.3 mm particle diameter).
Temperature of the injector: 190.degree. C.
N.sub.2 stream rate: 50 ml/min.
H.sub.2 stream rate: 50 ml/min.
Stream rate of the air: 200 ml/min.
Paper movement; 160 cm/hour.
Duration of chromatography: 20 min.
Sample volume: 1 .mu.l.
Triplicate measurements are made from each sample. The standard solution
contains acetic acid, propionic acid, isobutyric acid, butyric acid,
isovaleric acid and valeric acid.
More than 90 strains of bacteria were isolated from a sheep fed on a
complete ration. Then the strains were labelled genetically and examined
(the positive strains were examined several times). Representative results
are shown in Table 8.
Explanation of the signs used in Table 8:
S: inoculated after sterilization
NS: culture containing living rumen flora was inoculated
a) trace amounts;
b) negative control: volatile fatty acid content of media prepared from
rumen liquor, physiological buffer and feed used for the experiment
(average of 12 measurements;
c) positive control: volatile fatty acid content of the incubated culture
containing the initial rumen bacteria and otherwise prepared by the same
process (average of 12 measurements);
d) as c) but 5 ppm monensin Na were added to the media (average of 6
determinations);
e) as c), but 10 ppm monensin Na was added to the media (average of 6
determinations).
TABLE 8
__________________________________________________________________________
Bacterium
Re-
Acetic acid
Propionic acid
i-Butyric acid
Butyric acid
i-Valeric acid
Valeric
Acetic acid/
strain mark
.mu.g/ml
.mu.g/ml
.mu.g/ml
.mu.g/ml
.mu.g/ml
.mu.g/ml
propionic
__________________________________________________________________________
acid
Hh-GYOKI-
S 1,71 1,87 a 0,35 a -- 0,91
1-123Sz NS 2,00 2,60 a 0,50 a -- 0,77
Hh-GYOKI-
S 0,38 0,58 a 0,35 a -- 0,65
2-14Ab NS 1,95 1,36 a 0,52 0,12 a 1,43
Hh-GYOKI-
S 2,21 0,67 a a a -- 3,29
3-81Me NS 2,81 0,81 a a a -- 3,47
b Con-
1,51 0,74 a 0,39 a a 2,04
c trol
1,68 0,91 a 0,51 0,11 0,09 1,84
d 1,69 1,08 a 0,41 a a 1,67
e 1,61 0,91 a 0,37 a a 1,77
Hh-GYOKI-48a
S 1,26 1,60 a 0,32 a -- 0,79
NS 1,21 2,38 a 0,34 0,48 a 0,51
Hh-GYOKI-50a
S 1,44 1,56 a 0,34 a -- 0,92
NS 1,73 2,01 a 0,35 0,15 -- 0,86
Hh-GYOKI-51a
S 2,37 0,47 a 0,36 a -- 5,04
NS 2,02 0,70 a 0,35 0,15 -- 2,89
Hh-GYOKI-55a
S 2,10 0,51 a 0,34 a -- 4,12
NS 2,18 0,78 a 0,37 0,14 -- 2,79
Hh-GYOKI-113
S 1,39 0,67 a 0,34 -- -- 2,07
NS 1,77 0,60 a 0,27 -- -- 2,95
Hh-GYOKI-122
S 1,31 0,87 a 0,31 -- -- 1,50
NS 2,02 0,91 a 0,31 -- -- 2,22
Hh-GYOKI-109b
S 2,49 0,45 a 0,29 -- -- 5,53
NS 2,92 0,36 a 0,30 -- -- 8,11
Hh-GYOKI-126
S 0,56 0,60 a 0,52 a a 0,93
NS 0,83 1,14 a 1,55 0,11 0,70 0,73
__________________________________________________________________________
The data of Table 8 indicate that the ratios of volatile fatty acids
produced by the fermentative function of the rumen flora can be modulated
in a wide range by the administration of microbial cultures prepared
according to the invention. E.g. production of propionic acid can be
significantly stimulated with a culture prepared from strain Hh-GYOKI-48a,
while strain Hh-GYOKI-109b stimulates production of acetic acid.
Stimulation of production of individual fatty acids was observed both on
media lacking (S) or containing (NS) living rumen microbes. In our
experimental system monensin Na decreased the ratio of acetic acid to
propionic acid by 0.1 or 0.2 (d, e).
Microorganisms chosen by the above-mentioned process are labelled
genetically, administered to ruminants and examined for ruminal growth and
persistence by repeating the process described in item B) of Example 1.
Strains with an advantageous fermentative pattern and long ruminal
persistence will be orally administered for modifying the production of
volatile fatty acids.
EXAMPLE 5
Bacterial preparation for oral administration to ruminants
Bacteria to be administered are cultured on RGCA+CG media (Example 4) under
anaerobic conditions, by the described process. After cultivation, the
cells are separated by filtration or centrifugation. Separated cells are
suspended in physiological buffer (Example 4) and freeze dried. The
lyophilized bacterial preparation is stored, suitably formulated and
administered to ruminants orally.
Microorganisms may be cultivated in other conventionally used media as
well, e.g. in media containing glucose and starch etc. as carbon source
and inorganic salts as N-source.
The preparation can be easily administered by mixing it to feed or drinking
water, alone or together with other biologically active agents, e.g. with
antibiotics and vitamins.
In addition to the freeze-dried preparation other products can be prepared
as well. The microorganisms may also be administered after mixing the
filtered or centrifuged bacterial mass with suitable carrier or diluting
substances, e.g. CaCO.sub.3, concentrates, premixes or other feedstuffs.
The bacterial strain(s) are chosen from the microorganisms, prepared by the
process of the invention and advantageously modifying the rumen flora, and
their quantity to be fed is determined depending on the ration and the use
of the animal. If a decrease of the acetic acid to propionic acid ratio is
required, we may use e.g. a culture prepared from strain Hh-GYOKI-48a, but
for an increase of the ratio the administration of strain Hh-GYOKI-3-81Me
is recommended.
Determination of the required microbial cell number may not mean any
difficulty for those skilled in the art. It is recommended to administer
the cells in a quantity to make 5.times.10.sup.2 to 5.times.10.sup.7
cultivated microorganisms per ml of rumen liquor.
EXAMPLE 6
Administration of strains Hh-GYOKI-48a and Hh-GYOKI-1-123Sz to sheep
Hh-GYOKI-48a strain is cultivated on RGCA"CG media (Example 4) in two
5-liter fermentors (useful volume) at 37.degree. C., under anaerobic
conditions. Fermentation is commenced by inoculation with a 10 ml culture
of similar composition. After 48 hours of cultivation the cells are
separated by centrifugation (5000 r.p.m.) and the wet sediment weighing 58
g is mixed carefully with 4 kg of corn meal. The mixture is divided to
eight equal parts and orally administered to eight sheep previously
starved for 24 hours. Strain Hh-GYOKI-1-123Sz may be used similarly, with
a bacterial harvest of 53 g.
In a growing-fattening experiment 23 sheep were ad libitum fed on poor
grass hay and the animals were weighted every week for 5 weeks. The
experimental groups consisting of eight sheep were fed by one of the
bacterial preparations each for a single feeding and seven sheep served as
control. 600 to 900 g of hay were consumed per day and animal, plus
mineral and vitamin premix mixed with corn meal (100 g.). The results are
shown in Table 9.
TABLE 9
______________________________________
Weight gain in sheep fed ad libitum on grass hay
Serial Initial 1st 2nd 3rd 4th 5th
number weight week
______________________________________
Control
1 29.0 29.5 30.0 29.5 29.5 29.5
2 27.0 27.5 28.5 27.0 26.5 28.0
3 28.5 27.0 27.0 27.5 26.5 26.5
4 30.0 30.5 30.0 30.5 30.0 29.0
5 29.5 30.0 29.0 30.5 29.5 30.0
6 25.0 25.5 26.5 26.0 26.0 27.5
7 27.0 27.0 27.5 28.0 28.0 28.0
Treated with strain Hh-GYOKI-1-123Sz
8 29.5 32.0 32.5 33.0 33.5 34.0
9 25.5 26.5 27.0 28.5 29.5 29.0
10 25.0 25.5 25.0 28.0 29.0 28.5
11 25.5 24.5 25.5 27.5 27.0 28.0
12 29.0 28.0 29.0 28.5 29.0 30.0
13 29.5 30.0 30.5 29.5 29.5 30.5
14 29.5 28.5 29.5 30.0 30.5 31.0
15 28.5 29.0 30.5 30.0 30.5 32.0
Treated with strain Hh-GYOKI-48a
16 29.5 30.0 30.5 31.0 32.0 33.5
17 26.5 25.0 26.5 27.0 29.0 30.0
18 27.0 27.5 28.5 29.5 30.5 31.0
19 26.0 26.0 27.0 28.0 29.0 30.5
20 29.5 30.5 31.0 31.5 32.5 33.0
21 28.0 28.0 28.0 29.0 29.0 30.0
22 29.0 30.5 33.2 30.0 32.8 33.5
23 27.0 26.0 27.5 29.0 31.0 32.0
______________________________________
The average daily weight gain is calculated from the date of Table 9 and
are shown in Table 10.
TABLE 10
______________________________________
Average weight and daily weight gain of
experimental and control sheep
Initial
1st 2nd 3rd 4th 5th
weight
week
______________________________________
Control
Mean body 28.00 28.14 28.36 28.43 28.00 28.36
weight (kg)
Mean daily +20 +31 +10 -61 +51
weight gain (g)
Treated with strain Hh-GYOKI-1-123Sz
Mean body 27.75 28.00 28.69 29.31 29.81 30.37
weight (kg)
Mean daily +31 +86 +77 +62 +70
weight gain (g)
Treated with strain Hh-GYOKI-48a
Mean body 27.81 27.94 29.02 29.37 30.73 31.69
weight (kg)
Mean daily +16 +135 +44 +170 +120
weight gain (g)
______________________________________
Sheep treated with strains Hh-GYOKI-1-123Sz and Hh-GYOKI-48a and the
control group gained on the poor ration in averge 2620, 3875 and 360 g,
respectively, during the 35-day experimental period.
Initial body weights did not differ significantly between groups, but
significant differences were found in the final body weights (Table 11)
and in the daily gains (Table 12).
TABLE 11
______________________________________
Statistical evaluation of final body weights
Control Hh-GYOKI-1-123Sz
Hh-GYOKI-48a
______________________________________
Mean (kg)
28.357 30.375 31.6875
Corrected
1.476 3.910 2.281
quadrate of
standard
deviation
p (%) <5.0 <0.1
______________________________________
TABLE 12
______________________________________
Statistical evaluation of body weight gains
(5 weeks)
Control
Hh-GYOKI-1-123Sz
Hh-GYOKI-48a
______________________________________
Number of 7 8 8
animals
Total weight
2.5 21.0 31.0
gain of the
group (kg)
Maximum gain
2.5 4.5 5.0
(kg)
Minimum gain
-2.0 1.0 2.0
(kg)
Mean gain per
0.3571 2.6250 3.8750
sheep (kg)
Corrected 2.143 1.768 0.839
quadrate of
standard
deviation
Standard .+-.1.355
.+-.1.244 .+-.0.857
deviation
______________________________________
The data indicate that preparations made according to the invention may
markedly stimulate weight gain in sheep.
EXAMPLE 9
Persistance of genetically labelled bacteria in the bovine rumen
The process described under item C) of Example 1 is repeated with the
difference that the strain Hh-GYOKI-1-123Sz resistant to 10,000 .mu.g/ml
kanamycin is cultivated in 4 liter of RGCFa media. After reaching the
stationary phase (38th hour) the culture is harvested by centrifugation
(5000 r.p.m.) and the cells thoroughly mixed with 500 g of corn meal are
fed to a cow. Weekly samples are taken through a fistula, and ruminal
persistence of the strain administered is determined according to item C)
of Example 1.
Results indicate that strain Hh-GYOKI-1-123Sz grows in the bovine rumen and
it can persist there for at least 40 days.
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